| Objective The aim of present study is to investigate the interactions at a metabolic level between doxofylline and the substrate of CYP450 fleroxacin in human liver microsomes and to account for the clinically observed interactions. The mechanism of the interaction between doxofylline and fleroxacin has also been investigated and the prediction of the potentiality of interaction in vivo by using the in vitro-in vivo extrapolation.Methods 1. Preliminary experiments were conducted to make sure all experiments were performed within the linear range of incubation time and protein concentration. The HPLC (High performance liquid chromatography) method to detect doxofylline was established.2. The HLM was pretreated with fleroxacin and then doxofylline was added. First, 50μg·mL-1 fleroxacin preincubated with HLM in various time (0-40min). Used the software Origin 7.5 to fit the IC50 of fleroxacin to CYP450. Second, fixed the concentration of fleroxacin, to investigate the influence of preincubation time to the quantity of the metabolite of doxofylline. The internal standard peak heights were expressed as a ratio to peak height of doxofylline for each concentration of inhibitor. These peak height ratios represented the remaining CYP450 activity in the HLM and were expressed as a percentage of the time-matched control samples without inhibitor.The natural logarithm of relative inhibition values were plotted versus preincubation time for a series concentration of fleroxacin used (1.25-100μg·mL-1), and the slopes determined by linear regression. These slope values represented the observed inactivation rate constants(kobs).Third, a double-reciprocal plot was then constructed using the reciprocal of the kobs (y-axis) and the reciprocal of the associated inhibitor concentration (x-axis)to estimated the values of kinact and KI, which were two principal kinetic constants that were specific for MBIs. They were the maximal rate constant of inactivation (kinact) and the inactivator concentration required for half-maximal inactivation (KI).3. Substituted the two parameters kinact and KI into equation (3) defined the fold reduction in intrinsic clearance (CLint) of a specific CYP caused by Mechanism-based inhibition.Results (1) A NADPH-dependent component was evident for fleroxacin, as the IC50 value was significantly reduced (i.e., more potent inhibition) when the inhibitor was preincubated with human liver microsomes. (2) With the increasing of concentration of fleroxacin, the inhibition became stronger. (3) At a fixed concentration the longer the preincubation time prolonged the stronger the inhibition became.(4) The time-,concentration- and NADPH-dependent characteristics confirmed the mechanism of fleroxacin inhibited CYP450 was mechanism -based inhibition, when doxofylline was the substrate of CYP450.(5) The Double-reciprocal plot of mean data points indicated a KI value of 6.754μmol·L-1,and a kinact value of 0.019min-1. The fold of reduction in CLint was 4.83-27.55, which was close to the result of in vivo study, the fold of AUC of doxofylline with the absence of fleroxacin increased 4.83-27.55. Thus, the possibility of toxity of doxofylline will be increased.Conclusions (1) Fleroxacin has strong inhibition on doxofylline of metabolism. (2) The main mechanism of fleroxacin and doxofylline in vivo interaction as the irreversible inhibition, fleroxacin inhibits cytochrome oxidase P-450 a subclass (CYP1A2), make the doxofylline metabolic disruption, doxofylline AUC increase 4.83-27.55 times, that of fleroxacin is a strong inhibitor of human liver microsomes CYP450 enzyme. (3). Fleroxacin have inhibition effects on metabolism of doxofylline in human s’ liver microsome. |
